Kit for co-purification and concentration of dna and proteins using isotachophoresis

ABSTRACT

A kit for separating and concentrating nucleic acid and protein targets includes labeled reagents which affect simultaneous co-purification and concentration of a nucleic acid and a protein, a gel isotachophoresis separation unit to which a sample comprising the nucleic acid and the protein is added, a detection unit for the detection of the presence of the nucleic acid and the protein, and instructions for use. The gel electrophoresis includes a gel box having a negative electrode side and a positive electrode side, the negative electrode side being filled with a first buffer comprising 2-Hydroxy-N-(tris(hydroxymethyl)methyl)-3-aminopropanesulfonic acid buffer and the positive electrode side being filled with a second buffer being different than the first buffer. The gel isotachophoresis separation is configured to subject the sample to isotachophoresis using a voltage.

CROSS REFERENCE TO RELATED APPLICATIONS

This application, is a divisional application of co-pending U.S. patentapplication Ser. No. 12/817,288, filed Jun. 17, 2010, which claimspriority to and the benefit of U.S. provisional application Ser. Nos.61/268,969 and 61/248,988, respectively filed on Jun. 18, 2009 and Oct.6, 2009, and which is also a continuation-in-part of U.S. patentapplication Ser. No. 12/808,023, filed Jun. 14, 2010 and issued on Dec.24, 2013 as U.S. Pat. No. 8,614,059, which is a §371 national stageentry of PCT/US08/65260, with an international filing date of Jul. 18,2008, and which claims priority to and the benefit of U.S. provisionalapplication Ser. Nos. 61/013,774 and 61/027,518, respectively filed onDec. 14, 2007 and Feb. 11, 2008, the entire content of all of which isincorporated herein by reference.

STATEMENT OF GOVERNMENTAL INTEREST

This invention was made with government support under contract number2007-st-061-000003-02 awarded by the Department of Homeland Security.The government has certain rights in the invention

BACKGROUND

This invention relates to a method of purifying proteins and nucleicacids

One of the largest contributors to false positive and false negativeresults in rapid detection technologies is inconsistent, variableprocessed samples. Sample preparation represents a critical, yet underdeveloped capability. Present day sample preparation is usuallyperformed manually. When automated, sample preparation involvesmechanizing manual processing methods resulting in large, complex,robotic systems with significant reagent/waste streams and consumableusage.

Generic methods for the purification of DNA from cells or mixtures ofcells have been available for many years and include alcoholprecipitation, silica binding, standard gel electrophoresis methods, andphenol/chloroform extraction. The polymerase chain reaction (PCR),through its use of primers to amplify and detect regions of thepreviously-purified nucleic acid specific for different classes oforganisms. The ability to develop generic purification methods fornucleic acids is due largely to the fact that all nucleic acid moleculesare similar in chemical structure and these similarities can be takenadvantage of when developing purification methods. However, most ofthese methods are time consuming and must currently be performedmanually.

Proteins, on the other hand, are very different in structure from onetype of protein to the next. Therefore, protein purification has largelyfocused on separation methods based on unique protein characteristicssuch as differences in size, charge, hydrophobicity, isoelectric point,antibody binding and/or enzyme-substrate specificities. When a number ofthese methods are performed in tandem eventually leads to a protein thathas been purified away from all other proteins in the starting mixture.Typically, separation methods include chromatography electrophoresis,immunoprecipitation and magnetic separation techniques and the like.Chromatography is performed using columns which are typically quitelarge and require expensive equipment to obtain and analyze the samples.Therefore, methods currently used to purify nucleic acids are not usedto purify protein and conversely, methods used to purify protein are notused purify nucleic acids.

U.S. Pat. No. 7,473,551 B2 (Warthoe) includes a microsensor and ahydrogel to isolate target analytes using a non-fluorescent detectionsystem, which includes the target analytes as being nucleic acids orproteins. U.S. Pat. No. 7,371,533 discusses a method of separatingpolypeptides or polynucleotides using electrophoresis. U.S. ApplicationNo. 2009/0178929 (Broer et al.) discusses a device for isoelectricfocusing for the separation of DNA or proteins. U.S. Application No.2009/0139867 A1 (Marziali and Whitehead) discusses methods and anapparatus for concentrating RNA or DNA. U.S. Application No.2008/0237044 A1 (Fiering and Keegan) discusses a method and apparatusfor continuously separating or concentrating molecules that includeflowing two fluids in laminar flow through an electrical field andcapturing at one of three outputs a fluid stream having a differentconcentration of molecules. Isotachophoresis is not disclosed in any ofthe applications, nor is the use of TAPSO buffer.

Isotachophoresis is a technique used to separate charged particles usinga discontinuous electrical field to create sharp boundaries between thesample constituents. In this method, the sample is introduced between afast leading electrolyte and a slow moving electrolyte to create awindow between which a subset of constituents from a complex matrix canbe segregated from other matrix constituents. The segregation occursafter application of an electrical field to the sample and theelectrolytes are allowed to separate by charge. The electrolytes usedfor a separation are selected experimentally so that as manycontaminating constituents as possible are excluded from the finalsample.

Isotachophoresis is currently used in the field of protein purificationas one potential method in capillary electrophoresis for the separationof specific proteins from a mixture of proteins. However, onedisadvantage of isotachophoresis in capillary use is that one can onlypurify either negative or positively charged ions in the capillary tubesbecause these moieties will migrate in different directions uponapplication of the electrical field. The most common use ofisotachophoresis is in protein stacking gels where protein samples areadded to a gel in a wide band for separation by molecular weight.However, before separation by molecular weight can occur, the proteinmust be concentrated into a small band to increase resolution.

U.S. Pat. No. 3,869,365 (Sunden) is drawn to a broad method ofcounter-flow isotachophoresis comprising two electrolytes and the flowand voltage adjusted to maintain the sample at a desired position in thecolumn. U.S. Pat. No. 3,948,753 (Arlinger) is drawn to an apparatus forisotachophoresis comprising a column, capillary tube, a detector, and ashunt tube bifurcating the column. U.S. Pat. No. 6,685,813 B2 (Williamset al.) is drawn to a method of separating components usingisotachophoresis. U.S. Appl. No. 2004/0060821 A1 (Williams et al.) isdrawn to a method of separating components using isotachophoresis. U.S.Pat. No. 7,494,577 B2 and U.S. Application No. 2004/0060821 (Williams etal.) discusses a method of separating components by loading amicrochannel with a sample, placed between a trailing-edge electrolyteand a leading-edge electrolyte using isotachophoresis. There is nodisclosure of the separation of either nucleic acids or proteins or to aTAPSO buffer. Application No. WO 2008/025806 (Gerhard Weber) is directedto a method of separating particles using electrophoresis and disclosesisotachophoresis. Baumann, G. and Chrambach, A. disclosed the use ofisotachophoresis for the isolation of hormones. (Proc. Natl. Acad. Sci.USA, 73(3): 732-736, (1976)). Böttcher, A. et al. disclosed preparativeisotachophoresis for separation of human plasma lipoproteins,apolipoproteins and HDL subfractions, (J. Lipid Res. 41:905-915,(2000)). None disclose the simultaneous separation and purification ofboth nucleic acids and proteins. U.S. Appl. No. 2004/0031683 A1 (Eipelet al.) is drawn to a method of fractionating proteins using severalprocedures, including isotachophoresis. The two-step process includescapillary electrophoresis, but does not disclose the separation ofnucleic acids. U.S. Pat. No. 7,399,394 B2 (Gerhard Weber) is directed toa free flow electrophoresis (FFE) (also known as carrierlesselectrophoresis or isotachophoressis) method and related devices. U.S.Pat. No. 5,817,225 (Hinton) is drawn to an electrophoresis unitcomprising an anode compartment, a cathode compartment, a separationchamber and an electrolyte in said chamber with an electrophoreticmobility between the mobilities of the nucleic acids and organic saltswhich are to be separated. The separation of proteins was not disclosed.U.S. Pat. Nos. 7,316,771 B2 and 7,247,224 (withdrawn) (both by GerhardWeber) is directed to a medium for electrophoresis comprising at leasttwo acids and at least two bases as buffers. Neither disclose theseparation of nucleic acids or proteins. U.S. Pat. No. 6,780,584 B1(Edman et al.) is broadly drawn to a device with a first bufferreservoir containing a first buffer with a differing conductances, aconductive semipermeable matrix, and a first and second electrode and aspecific binding entity and discloses a device which separates nucleicacid and RNA in hybridization reactions, but not the separation ofproteins. U.S. Pat. No. 5,464,515 (Bellon) is drawn to a procedure forloading one of several biological samples on an electrophoresis slabsupport, but does not disclose the separation of nucleic acids. U.S.Pat. No. 7,214,299 B2 (Armstrong) is broadly directed to the separationof microbes and cells using electrophoresis methods. U.S. Pat. No.5,447,612 (Bier et al.) is directed to a buffering composition forelectrophoresis methods, including TAPSO and EACA (epsilon-aminocaproicacid) as complementary buffer pairs and methods. comprising the buffercomposition in an isoelectric focusing method, used with a recyclingfocusing instrument of the '169 Bier patent. 2008/0197019 A1 (Santiagoand Khurana) discloses a method of using an electric field to isolateproteins and nucleic acids. Isotachophoresis is not disclosed, nor isthe use of TAPSO buffer. 2010/0029915 discloses automated methods toisolate proteins or nucleic acids comprising the use of BES buffer anddoes not mention ITP. Xu, Z Q et al., (J. Chromatography, A,1216(4):659-______, (January 2009)) discusses electrokineticsupercharging as a transient ITP including the separation of proteinsand nucleic acids.

Appl. No. EP 05076569.2 (Stichting voor de Technische Wetenschnappen) isdrawn to a device for separating particles and the use ofisotachophoresis for the non-simultaneous separation of nucleic acids orproteins and the use of binding molecules. Blessum, C. et al. disclosecapillary electrophoresis in the separation of proteins, nucleic acidsand lipoproteins, and its use in isotachophoresis. (Ann. BiologieClinique, 57(6):643-647 (1999) [French]). Blessum et al. do not disclosethe simultaneous isolation of nucleic acids and proteins usingisotachophoresis. Dolnik, V. et al. disclose capillary electrophoresistechniques and microchip technology which includes isotachophoresis, andcurrent methods of separation of nucleic acids or proteins, but does notdisclose simultaneous separation of nucleic acids and proteins(Electrophoresis, 21(1):41-54, (1999) [Abstract only]). PCT ApplicationNo. WO 2008/082876 A1 (Balgley) is directed to a method of separatingDNA or protein from a heterogeneous biomolecular sample usingisotachophoresis coupled with liquid column chromatography. PCTApplication No. WO 2008/053047 A2 (Weber, Gerhard) is directed to anelectrophoresis method comprising a spacer zone and also separating atleast one analyte, including DNA, protein or protein complexes usingisotachophoresis. PCT Application No. WO 2007/008064 A1 (Kohlheyer etal.) is directed to a device and use of the device for separatingparticles in a fluid sample utilizing free flow isotachophoresis; and adiscussion of the separation of DNA and proteins, although there is nomention of simultaneous separation. U.S. Pat. No. 4,897,169 (Bier andTwitty) discloses isotachophoresis and an apparatus using TRIS andcacodylic acid as electrolyte components. Hirukawa, T. et al. discussesthe use of ITP for the separation of DNA fragments and SDS-proteins, butnot simultaneously. (Bunseki Kagaku, (Japanese language, Abstract andFIGS. 13 and 16) 52(12):1069-1079 (2003)). Khurana, T. K. et al.describe ITP separation of DNA and proteins (Lab Chip, 9:1377-1384(March 2009)). U.S. Patent Publication No. 2009/0032401 A1 (Ronaghi,Khurana, and Santiago) discloses a method of using an electric field toisolate proteins and nucleic acids, concentrating and separating atleast one directly undetectable analyte of interest and said at leasttwo directly detectable spacer molecules into zones usingisotachophoresis; the analytes can be DNA or RNA. Lin, C-C et al.,discusses an integrated ITP-gel electrophoresis device and discusses theseparation of DNA and protein molecules. (Electrophoresis,29(6):1228-1236, (March 2008)). Shackman, J. G and Ross, D. discusses acapillary isotachophoresis method and discusses the separation of DNA orprotein molecules (Anal. Chem., 79(17):6641-6649, (August 2007)).

Newer, rapid, simpler methods of sample processing are required tosupport the next generation detection systems. The newer detectionmethods will have a heavier reliance on sample preparation for thegeneration of meaningful results. In addition, sample preparationmethods will need to support numerous different detection systems and becapable of being integrated as part of a complete sample processing anddetection system.

Therefore, there is currently a great need in sample processing methodsthat are fast, inexpensive, and are easy to perform, even by untrained,non-technical staff in a variety of disciplines including biodefense,food and water, agricultural, environmental, clinical testing, and thelike.

BRIEF SUMMARY

The present invention is drawn to a method of simultaneouslyco-purifying and concentrating nucleic acid and protein targets into asingle volume that can then be tested on a variety of sensortechnologies. This method represents a simple system that is readilyautomated into a hand held, disposable device capable of being operatedby unskilled operators in a field environment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a standard 1% agarose submarine gel showing the relativemobility of genomic DNA isolated from B. atrophaeus (BG) and E.herbicola (EH).

FIG. 2 shows a standard 1% agarose isotachophoresis gel showing therelative mobility of genomic DNA isolated from B. atrophaeus and E.herbicola.

FIG. 3 shows a standard 1% agarose submarine gel showing the relativemobility of purified proteins.

FIG. 4 depicts a standard 1% agarose isotachophoresis gel showing therelative mobility of purified proteins.

FIG. 5 shows an isotachophoresis gel prior to the application of voltageand showing the DNA, protein, xylene cyanol and humic acid combined andlocated within a single sample well and showing the dark color of thehumic acids.

FIG. 6 demonstrates an isotachophoresis gel after the completion of therun showing the separated DNA, protein, and xylene cyanol dyeconcentrated into a thin band (blue) separated from the humic acids(brown) which are not concentrated by the system.

FIG. 7 is a graphic representation of Ct values of PCR reactionscomparing the relative amplification of BG and EH DNA dilutions fromsamples containing humic acid and sample containing humic acid purifiedby isotachophoresis.

FIG. 8 shows the Ct values of PCR reactions comparing the relativeamplification of BG and EH DNA dilutions from samples containing humicacid and sample containing humic acid purified by isotachophoresis.

FIG. 9 depicts a 1% agarose isotachophoresis gel showing high and lowmolecular weight DNA ladders, and calf thymus DNA.

FIG. 10 depicts a 1% agarose isotachophoresis gel showing high and lowmolecular weight DNA ladders, and calf thymus DNA.

FIG. 11 shows a 1% isotachophoresis agarose gel run with proteins (leftpanel A) and DNA (right panel B). The gel was run using TAPSONaOH/Bis-Tris MES/Acetic acid.

FIG. 12 shows a 1% istotachophoresis agarose gel run with proteins (leftpanel A) and DNA (right panel B). The gel was run using TAPSONaOH/Bis-Tris MES/Acetic acid followed by the removal of TAPSO/NaOH andits replacement with Bicine in the buffer chamber, resulting in twobuffer fronts.

FIG. 13 depicts an isotachophoresis gel showing nucleic acids andproteins from a 293FT human cell extract and purified RNA from the sameextract.

DETAILED DESCRIPTION

The present invention is drawn to a method of simultaneouslyco-purifying and concentrating nucleic acid and protein targets into asingle volume. The entire sample preparation process, regardless of thedetection method can be automated using the invention. This automatedsample preparation method is capable of having an analyst add a sampleinto a device that performs all of the steps necessary to prepare asample for analysis. The present invention includes methods in whichsamples are not split during the sample preparation process and wherecommon purification methods can be used for purifying multiple analytes.The sample processing methods of the invention are fast, inexpensive,and are easy to perform, even by untrained, non-technical staff in avariety of disciplines including biodefense, food and water,agricultural, environmental, clinical testing, and the like.

“Nucleic acid” molecules, as used herein include DNA, RNA,polynucleotides and oligonucleotides; synthetic or naturally-occurring.The nucleic acid molecules include any single-stranded sequence ofnucleotide units connected by phosphodiester linkages, or anydouble-stranded sequences comprising two such complementarysingle-stranded sequences held together by hydrogen bonds. Unlessotherwise indicated, each nucleic acid sequence set forth herein ispresented as a sequence of deoxyribonucleotides (abbreviated A, G, C andT). However, the term “nucleic acid” includes a DNA molecule orpolynucleotide, a sequence of deoxyribonucleotides, or an RNA moleculeor polyribonucleotide. The corresponding sequence of ribonucleotidesincludes the bases A, G, C and U, where each thymidinedeoxyribonucleotide (T) in the specified deoxyribonucleotide sequence isreplaced by the ribonucleotide uridine (U).

Nucleic acids may originate in viral, bacterial, archobacterial,cyanobacterial, protozoan, eukaryotic, and/or prokaryotic sources. AllDNA provided herein are understood to include complementary strandsunless otherwise noted. It is understood that an oligonuleotide may beselected from either strand of the genomic or cDNA sequences.Furthermore, RNA equivalents can be prepared by substituting uracil forthymine, and are included in the scope of this definition, along withRNA copies of the DNA sequences isolated from cells or from virionparticles. The oligonucleotide of the invention can be modified by theaddition of peptides, labels, and other chemical moieties and areunderstood to be included in the scope of this definition.

Nucleic acid molecules detected or used by the methods or systems of theinvention may also include synthetic bases or analogs, including but notlimited to fluoropyrimidines, pyrimidines and purine nucleosideanalogues include, fluoropyrimidines, including 5-FU (5-fluorouracil),fluorodeoxyuridine, ftorafur, 5′-deoxyfluorouridine, UFT, carboranylthymidine analogues, FMAUMP(1-(2-deoxy-2-fluoro-D-arabinofuranosyl)-5-methyluracil-5′-monophosphate)and S-1 capecitabine; pyrimidine nucleosides, include deoxycytidine,cytosine arabinoside, cytarabine, azacitidine, 5-azacytosine,gencitabine, and 5 azacytosine-arabinoside; purine analogs include6-mercaptopurine, thioguanine, azathioprine, allopurinol, cladribine,fludarabine, pentostatin, 2-chloroadenosine, AZT, acyclovir,pencilcovir, famcyclovir, didehydrodideoxythymidine, dideoxycytidine,-SddC, ganciclovir, dideoxyinosine, and/or 6-thioguanosine, for example,or combinations thereof.

“Proteins” as used herein include peptides and polypeptides. A proteinis a large molecule composed of one or more chains ofnitrogen-containing amino acids linked together in a peptide linkage, ina specific order determined by the base sequence of nucleotides in theDNA coding for the protein. Examples of proteins include whole classesof important molecules, among them enzymes, hormones, antibodies andtoxins. Proteins as used herein are composed of 20 standard amino acids,or may contain synthetic or naturally occurring non-standard aminoacids. The amino acids present in the protein may be aromatic, D or Lconfiguration, modified or having an R or S chirality. Such proteins maycontain amino acids with posttranslational modifications. Suchposttranslational modifications include but are not limited tocarboxylation of glutamate, hydroxylation of proline or the addition oflong hydrophobic groups can cause a protein to bind to a phospholipidmembrane. Proteins may originate in viral, bacterial, archobacterial,cyanobacterial, protozoan, eukaryotic, and/or prokaryotic sources.

The proteins detected or used by the methods or systems of the inventioninclude polypeptides and/or peptides and further include proteins thatare part of a chimeric or fusion protein. Said chimeric proteins may bederived from species which include primates, including simian and human;rodentia, including rat and mouse; feline; bovine; ovine; including goatand sheep; canine; or porcine. Fusion proteins may include syntheticpeptide sequences, bifunctional antibodies, peptides linked withproteins from the above species, or with linker peptides. Polypeptidesof the invention may be further linked with detectable labels, metalcompounds, cofactors, chromatography separation tags or linkers, bloodstabilization moieties such as transferrin, or the like, therapeuticagents, and so forth. The proteins/peptides of the invention mayoriginate in viral, bacterial, archobacterial, cyanobacterial,protozoan, eukaryotic, and/or prokaryotic sources.

Antibodies detected or used by the methods or systems of the inventioninclude an antibody which is labeled with a labeling agent selected fromthe group consisting of an enzyme, fluorescent substance,chemiluminescent substance, horseradish peroxidase, alkalinephosphatase, biotin, avidin, electron dense substance, and radioisotope.The antibody of this invention may be a polyclonal antibody, amonoclonal antibody or said antibody may be chimeric or bifunctional, orpart of a fusion protein. The invention further includes a portion ofany antibody of this invention, including single chain, light chain,heavy chain, CDR, F(ab′)2, Fab, Fab′, Fv, sFv, dsFv and dAb, or anycombinations thereof.

The invention is further drawn to an antibody-immobilized insolublecarrier comprising any of the antibodies disclosed herein. Theantibody-immobilized insoluble carrier includes culture plates, culturetubes, test tubes, beads, spheres, filters, membranes, or affinitychromatography medium.

The invention also includes labeled antibodies, comprising a detectablesignal. The labeled antibodies of this invention are labeled with adetectable molecule, which includes an enzyme, a fluorescent substance,a chemiluminescent substance, horseradish peroxidase, alkalinephosphatase, biotin, avidin, an electron dense substance, and aradioisotope, or any combination thereof.

The method of separation of the present invention may include separationmedium including, agarose, polyacrylamide, mixed bed of agarose andpolyacrylamide, starch, acrylamide-urea, and/or other separation mediaknown in the art, in liquid, gel or membrane form, or a combinationthereof. The separation may be done in slab electrophoresis units,columns, tubes, glass plates or slides, wells, membranes or the like.

At the present time, there are no rapid sample preparation methods,either manual or automated, that have demonstrated co-purification andconcentration of both immunoassay protein targets and nucleic acid PCRtargets in a single output.

One embodiment of the invention is a method and apparatus tosimultaneously co-purify and concentrate nucleic acid and proteintargets into a single volume that can then be tested on a variety ofsensor technologies. In addition, this method represents a simple systemthat can be readily automated into a hand held, disposable devicecapable of being operated by unskilled operators in a field environment.The invention allows the purification of both nucleic acid and proteinsimultaneously using isotachophoresis. The sample is added to the middleof a device that allows isotachophoresis to occur in two directionstoward both the positive and negative electrodes when a voltage isapplied. A buffer system provides for “window DNA” moving toward thepositive electrode and “windows” protein moving toward the negativeelectrode. Buffer conditions are chosen so that all proteins of interestwould be positively charged to separate them from the DNA. If possible,the buffers are chosen to separate the proteins of interest from othercontaminating proteins to increase the purity of the protein samplefurther.

Another aspect of the invention includes isotachophoretic purificationand concentration of bacteria and viruses from complex samples. In manycases, the infectious dose of a bacteria or virus maybe as little as asingle organism and that single organism may be present in a largevolume of matrix. Because biological detectors can only test very smallvolumes, the typical strategy is to develop sampling plans in which onlya statistical subset of each sample is tested. Therefore, a negativeresult only demonstrates that the sample subset that is tested isnegative and does not mean that the entire sample is negative. Thepresent invention allows a greater statistical probability that anentire sample is truly negative. The probability can be increased bytesting more of the sample. Since the methods of the inventionconcentrate the organisms or target moieties present in a sample fromthe larger volume to a smaller volume, the probability of detectionincreases. Since bacterial cells and viruses have a net charge, theywill also move in an electrical field. Therefore bacteria and virusescan be concentrated using the isotachophoretic method as describedherein for nucleic acids and proteins. Since the isotachophoreticseparation of bacteria and viruses or proteins and nucleic acids alsopurifies them from matrix components that could affect assay results,the resulting purification also allows more reproducible testing bymolecular and immunoassays. It is envisioned that a system of theinvention includes one or more cartridges that all run on a single,portable instrument. These cartridges could be disposable, re-usable orintegrated into the system; and optionally could be interchangeable. Toseparate and concentrate bacteria and viruses in a sample matrix onedisposable cartridge could be used; and the separation and purificationof nucleic acids and protein, a different cartridge could be used.

The gold standard method for the detection of bacterial or viralpathogens remains culture-based even these methods are time consuming,typically requiring 1-2 days to make a final determination. A vastnumber of new, rapid detection technologies are poised to revolutionizethe field of pathogen detection by delivering results in less than onehour, but they have been limited by significant false positive and falsenegative rates. One potential solution to decrease the numbers of falseresults generated is to fuse multiple disparate sensing technologiesthat provide truly orthogonal sensing capabilities. This type ofapproach is currently employed in a few cases where organisms aredifficult or impossible to grow such as the detection of HCV virus inclinical settings where both immunoassay and PCR test results are usedcooperatively in diagnosing disease. This approach has remainedlaboratory based due to the size and complexity of the equipmentrequired for rapid detection approaches. However, more rapid and moresensitive technologies are currently being developed. A new generationof instruments will allow a paradigm shift in the way clinical andenvironmental diagnostics are performed because the technologies can becarried to the sample for testing as opposed to collecting the sampleand taking it to the laboratory.

Sample preparation represents a critical, yet under-developedcapability. One of the largest contributors to false positive and falsenegative results in rapid detection technologies is inconsistent,variably-processed samples. Therefore, there is a great need for thedevelopment of automated, rapid, reliable, and reproducible samplepreparation methods. In most cases, present day sample preparation isperformed manually and automation of sample preparation has involvedmechanizing manual processing methods resulting in large, complex,robotic systems with significant reagent/waste streams and consumableusage. The present invention represents a newer, rapid, and simplermethod of sample processing to support the next generation of detectionsystems which have an even heavier reliance on sample preparation forthe generation of meaningful results. In addition, sample preparationmethods of the invention is capable of standing alone to supportnumerous different detection systems and also is capable of beingintegrated as part of a complete sample processing and detection system.Sample preparation methods of the invention are also capable ofpurifying multiple analytes for detection to support an orthogonalsensing approach. The present invention avoids splitting a sampleprocess on two different instrument systems, which can raise a number ofissues regarding chain of custody requirements and sample consistency.

Detection methods for proteins include, but are not limited to:immunoassay, protein sequencing, mass spectrometry, functional assaysthat detect activity (such as enzymatic or protease assays), ligandbinding but without the use antibodies, such as specific bindingreceptors, aptamers, gels, and/or combinations thereof. Detectionmethods for nucleic acids include, but are nucleotide limited to: PCR,isothermal amplification methods, hybridization reactions, microarrays,protein-DNA binding, mass spectrometry, gels, and/or combinationsthereof. Oligonucleotides used in the systematic evolution of ligands byexponential enrichment (SELEX) are obtained from a random sequencelibrary obtained from a combinatorial chemical synthesis of DNA. Theoligonucleotide library is then screened for specific binding to targetsequences for use as aptomers (U.S. Pat. Nos. 5,637,459, 5,475,096 and5,270,163, all incorporated herein by reference)..

In addition to sample preparation playing a vital role in thereproducibility of test results, the sample preparation methods of thepresent invention are applied to concentrate samples from large volumes.Sample concentration is an important step because most present daybiological sensors require input volumes of less than 100 μL with PCRtests requiring as little as 1 μL. Microfluidic and chip-basedapproaches require sample sizes to be reduced even further into thesubmicron range. This reduction in input volume will result in areduction in overall test sensitivity if analyte is not concentratedduring the sample preparation procedure.

Another aspect of the invention is used in an application whereconcentration of all nucleic acids from a sample into a single aliquotwould not be advantageous. Without being limited, the method of theinvention can be used for nucleic acid sequence analysis of samples. Itis anticipated that the invention can be used for the rapididentification of nucleic acid sequences, including, but not limited tobacterial or viral nucleic acid sequencing. It is anticipated that theinventive method will become the method of choice for rapididentification of bacteria and viruses from both clinical andenvironmental samples.

Traditionally, nucleic acid sequencing is a shotgun method whereby allnucleic acids in a sample are analyzed simultaneously regardless of theorigin. Therefore, the most abundant types of nucleic acids in a samplewill be identified and less abundant nucleic acids will be masked. Inmany types of samples it is this less abundant nucleic acids that arecritical for identifying pathogens of interest. One of the biggestcontributing factors for DNA that is present in large concentrations(masking DNA) is from human cells that are present in large quantitiesin many clinical sample types. The invention overcomes these drawbacksin traditional nucleic acid sequencing by removing human nucleic acidsfrom a sample while purifying and concentrating bacterial and viralnucleic acids, is a significant step toward the application of nucleicacid sequencing and similar methods as a diagnostic tool. Under currentsystems, mitochondrial DNA comigrates with viral and bacterial nucleicacids. The method and apparatus of the invention includes the separationof mitochondrial DNA from bacterial and viral nucleic acids. In theisotachophoresis system of the invention, bacterial and viral nucleicacids are both purified and concentrated. However, small nucleic acidsare not concentrated with the bacterial and viral nucleic acids becausethey migrate in the gel faster than the leading edge of the fast movingelectrolyte. Therefore, while the method separates most nucleic acids byisotachophoresis, for small nucleic acids, there is a size separationcomponent as well. Size is also a critical difference between humangenomic nucleic acids and bacterial/viral nucleic acids in that humangenomic DNA is much larger. The invention takes advantage of this sizedifference in the gel-based isotachophoresis format to simultaneouslyconcentrate bacterial and viral nucleic acids while excluding humangenomic nucleic acids based on size. One of the most important factorscontrolling the degree of migration possible in a gel electrophoresisformat is the amount of cross linking in the gel, which is directlyrelated to the concentration of agarose. Therefore, the cross-linking ofthe gel is set such that migration of large nucleic acids in the gel isprevented, but that smaller nucleic acid molecules are still able toenter the gel. Thus, bacterial and viral nucleic acids are purified andconcentrated, while at the same time the bacterial and viral nucleicacids are separated from genomic nucleic acids.

Another aspect of the invention is a method, apparatus and kit whichallows both DNA and protein to be concentrated separately so that eachcan be isolated independently. Additionally, the invention includes amethod, apparatus and kit which allows for the simultaneous andindependent isolation of species of proteins to be isolatedsimultaneously and independently from different species of nucleicacids.

Kits of the invention would include labeled reagents and instructionsfor use of such reagents either in combination with an includedapparatus or with use of a separate apparatus.

WORKING EXAMPLES

Buffer Abbreviations:

Bicine buffer, (N,N-Bis(2-hydroxyethyl)glycine),Bis-Tris(2-(bis(2-hydroxyethyl)amino)-2-(hydroxymethyl)propane-1,3-diol),TAPSO (2-Hydroxy-N-(tris(hydroxymethyl)methyl)-3-aminopropanesulfonicacid), HEPES (4-(2-hydroxyethyl)-I-piperazine ethanesulfonic acid), MES(2-(N-morpholino)ethanesulfonic acid).

Example 1 Purification and Characterization of Nucleic Acid Methods:

Bacillus atrophaeus (BG) spores were prepared by adding 1 gram of alyophilized spore stock to 1 mL of ddH2O. Erwinia herbicola (EH) cellswere prepared by growing cells from a freezer stock to mid-log phase innutrient broth. Cells were harvested by centrifugation suspended inddH2O. BG spores and EH cells were lysed and their genomic DNA purifiedusing the QIAMP DNA Mini Kit (PN 56304, Qiagen) according tomanufacturer's instructions with minor modifications to the volumes andincubation times. Purified DNA was quantitated by OD260/280 ratios(Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual,3rd edition, Cold Spring Harbor Laboratory Press) and previouslyvalidated in-house real time polymerase chain reaction assays (RT-PCR)targeting the recA gene of BG and the chorismate mutase (aroQ) gene ofEH. Purified DNA was added directly to the standard humic acid solutionprior to separation. DNA was diluted to a concentration in which therewas not enough DNA present to reproducibly generate a positive PCRreaction (endpoint).

Characterization of Proteins

Proteins for this study were chosen to represent a broad range ofmolecular weights and isoelectric points. Purified proteins includingovalbumin, bovine serum albumin, horse radish peroxidase, and lysozymewere purchased from Sigma-Aldrich Corporation. All proteins were dilutedto standard concentrations of 1 mg/mL (wt/vol) in Bis-tris MES buffer.

Buffer Formulations

All buffers for the isotachophoresis experiments were prepared to finalconcentrations based on empirical experimental results as describedbelow. Higher or lower concentrations can be used for isotachophoresisbased on the amount of material being separated and the degree ofseparation required. Buffers were allowed to remain at the pH obtainedfrom the dilution of the material into double-distilled deionized water(ddH₂O).

Agarose Gel Electrophoresis

Standard submarine agarose gels were prepared using 1% Agarose MP (RocheApplied Science) in 0.5× tris-borate-EDTA buffer (Bio-Rad Laboratories).

Agarose Gel Isotachophoresis

Agarose gel electrophoresis was performed using a standard agarose gelelectrophoresis unit (Bio-Rad Laboratories) except the gel was run as asea level gel as opposed to a submerged gel. All gels were preparedusing 1% wt/vol MP agarose, because this was the lowest concentration ofagarose that provided a gel that was rigid enough to handle withouttearing. However, concentrations between 0.5-1.5% were tested anddetermined to be acceptable. Even broader ranges of agaroseconcentrations could be used as long as the agarose concentration washigh enough to form a crosslinked gel and low enough to allow migrationof large molecular weight bacterial genomic DNA into the gel.

For all isotachophoresis experiments, 1% agarose gels were preparedusing 0.4 M Bis-tris and 0.1 M MES. Gels were poured directly on thesurface of the electrophoresis unit as opposed to the gel tray that wasprovided. This allowed a complete seal on the sides and bottom of thegel to isolate the anode and cathode buffer chambers. The gel comb wasmodified by taping the teeth of the comb to form a single, long well toallow a larger sample volume ranging from 800 microliters (μL) to 1 mLto be separated. The comb was placed into the gel approximately 1 inchfrom the cathode edge. Two different buffers were used to fill anode andcathode buffer reservoirs. The cathode buffer consisted of 0.2 M Bicineand 0.1 M NaOH and the anode buffer consisted of 0.4 M BisTris and 0.1 Macetic acid. Buffer was added to the reservoirs so that they were evenwith the top of the gel so that the two buffer systems remainedseparated throughout the run. Gels were run at 150 volts forapproximately 4 hours for the nucleic acid and protein to beconcentrated and purified.

To extract the DNA from the gel, a long well was cut in the gel directlyin front of the dye line, using a razor blade. The well went all the wayto the bottom of the gel and was slightly longer than the dye line. Thewell was filled with the BisTris MES buffer. Current was again appliedto the gel and the dye moved into the cut well. Using a pipette, theliquid dye and DNA could be removed. This was normally done severaltimes in order to remove all of the dye/DNA.

A variety of different buffer formulations were used in a gel-basedisotachophoresis system to separate and concentrate nucleic acids andproteins. The combination that performed the best consisted of aBis-tris MES solution in the gel, a Bicine/NaOH cathode buffer and aBis-tris/acetic acid solution as the anode buffer. Therefore, thisbuffer system was used for further study into the feasibility of usingan isotachophoretic method for simultaneous purification of nucleic acidand proteins. However, it should be noted that the method is not limitedto these buffers alone and that further effort will yield buffer systemsthat perform similarly, or possibly even better. Therefore, the methodis not limited specifically to the buffer system chosen for theexperiments described below.

DNA isolated from Bacillus atrophaeus (BG) and Erwinia herbolitica (EH)was purified and then separated by submarine agarose gel electrophoresisin order to determine the size and quality of the genomic DNA that wasisolated (FIG. 1). In both instances genomic DNA can be observed in aband at a molecular weight of >10,000 base pairs with a few lighterbands at around 1000 base pairs. These lighter bands could representeither DNA from small plasmids present in the cell or genomic nucleicacids that have been degraded during the purification process. Molecularweight standards also separated on the gel according to their relativesizes as denoted FIG. 1 with larger pieces of DNA migrating lessdistance than smaller sized DNA. When the same isolated purified DNA wasrun on a sea level isotachophoretic gel, the large molecular weightbands of the molecular weight standards along with the BG and EH genomicDNA comigrated with the xylene cyanol dye between the leading edge ofthe Bicine buffer front (from the anode buffer chamber) and the trailingedge of the MES buffer (from the agarose gel; FIG. 2). DNA at molecularweights of approximately 2000 base pairs and below migrated through thegel at an equivalent or greater speed than the Bicine buffer front andtherefore, these smaller molecular weight DNA molecules separated basedon molecular weight and were not concentrated at the interface of theleading and trailing buffer components.

Purified proteins of various molecular weights and isoelectric pointswere purchased commercially and diluted to concentrations of 1 mg/mL inphosphate buffered saline. The proteins were then separated on a 1%agarose submarine gel with 0.5×TBE buffer which was identical to the gelused to separate DNA except for the proteins which were loaded in themiddle of the gel as opposed to loading near the anode end of the gel.As can be seen in FIG. 3, two of the four proteins, BSA and ovalbumin,migrated toward the cathode while the other two proteins, lysozyme andhorse radish peroxidase, migrated in the opposite direction toward theanode. Additionally, due to the fact that these proteins have mixedcharges at pH 8.0, they do not migrate as discrete bands, but smearthroughout the gel lane making characterization difficult. When the samefour proteins were loaded onto an isotachophoretic gel identical to thegel run for the purification of DNA (FIG. 4), three of the four proteinsincluding BSA, ovalbumin, and horse radish peroxidase all migratedtoward the cathode. In addition, the three proteins comigrated with thexylene cyanol dye between the leading edge of the Bicine buffer frontand the trailing edge of the MES buffer in the exact same position thatthe large molecular weight DNA and the xylene cyanol migrated in FIG. 2above. The fourth protein, horseradish peroxidase, presumably moved inthe opposite direction even in the isotachophoresis buffer system. Itwas not possible to move the protein starting locations to the middle ofthe gel in order to perform isotachophoresis due to the constraints ofthe buffer system, but had protein moved toward the cathode, it shouldhave been seen by the dye staining.

Once the relative mobilities of the bacterial genomic DNA and proteinswere established on the isotachophoretic gel, proteins, DNA, and xylenecyanol were mixed with a standard concentration of humic acids thatgenerated a reading of 1.0 at an optical density of 203.6 nm. Thismixture was then separated on an isotachophoretic gel under identicalconditions to the previous gels shown in FIGS. 2 and 4. Because themethod not only separates humic acids from both nucleic acids andprotein, but also concentrates nucleic acid and protein at a singlelocation on the gel, extremely wide loading positions can be cut toincrease the volume of material added to the gel. Increasing the volumeof material that can be separated will greatly enhance the sensitivityof any test run on the purified material. The relative positions of theprotein and large molecular weight DNAs were tracked according to theposition of the xylene cyanol dye and migration of the humic acids weretracked according to their characteristic visible brown color in thegel. FIG. 5 shows a typical isotachophoresis gel in which protein, DNA,xylene cyanol, and humic acids were mixed, prior to application ofvoltage; and FIG. 6 shows the same gel after separation of protein, DNA,xylene cyanol, and humic acids by isotachophoresis.

Humic acid is a known inhibitor of the polymerase that is used to copyDNA in the PCR reaction. Isotachophoretic purification of DNA from humicacids improves performance of PCR detection methods. Pure B. atrophaeusand E. herbicola DNA was combined with the standard concentration ofhumic acid to generate a single sample for analysis. A portion of thesample was removed to represent an unpurified sample and the remainingsolution was purified by isotachophoresis as described above. Sample wasrescued from the gel by first allowing separation to occur, turning offpower to the gel, and then cutting a small well into the gel between theleading edge of the DNA/xylene cyanol and the trailing edge of the humicacid. The newly created well was then filled with double distilleddeionized water prior to restoring power to the gel. Once power wasrestored, the xylene cyanol band was tracked until it entered the well,at which time power to the gel box was stopped again and the xylenecyanol and DNA was removed with a pipette. A comparison of xylene cyanolconcentrations based upon OD absorbance before and afterisotachophoretic separation was used to determine the percent recoveryof the sample. This percent recovery was then used to standardize theamount of solution added to the PCR reaction. Based on absorbancemeasurements, it was determined that recoveries of up to 95% with40-fold concentration were possible even in this gel box-based systemand even higher recoveries and greater sample concentration can beexpected as application specific electrophoresis units are employed.Both purified and unpurified DNA was then tested neat, with a 1:10dilution in ddH₂O, 1:100 dilution in ddH₂O, and a 1:1000 dilution inddH₂O. In addition, the BG DNA was further diluted to 1:10,000 in ddH₂Owhile EH DNA was not. The results (FIG. 8) show that the unpurified DNAwithout dilution could not be detected by PCR for either BG or EH due tofailure of the reaction to amplify even after 45 cycles. However, DNApurified by isotachophoresis could be amplified readily with Ct valuesof 18 for BG and 32 for EH. As samples are diluted, PCR reactioninhibitors such as humic acids are present at lower concentrationsallowing for more efficient amplification which would lead to lower CTvalues. However, the total amount of nucleic acid present is alsodiluted resulting in less nucleic acid to amplify, which leads to higherCT values. At some point, dilution of the humic acid inhibitor willreach the point that there is no longer inhibition of the polymerase andat that point, the Ct values of the purified and unpurified sample wouldbe expected to be the same. For the BG assay this occurs at the 1:10,000dilution and for the EH assay this occurs at the 1:000 dilution.

Purification

A mixture of Bacillus atrophaeus (BG) and Erwinia herho/itica (EH)genomic DNA; humic acid and purified proteins of various molecularweights and isoelectric points were separated on an isotachophoretic gelunder specific conditions (FIG. 6). The Figure shows the DNA, protein,and xylene cyanol dye concentrated into a thin blue band at a singlelocation on the gel, purified from the brown humic acids, which are notconcentrated by the system. The leveling bubble that can be seen underthe semi-transparent gel is not part of the actual separation process.

Recovery

The percent recovery of the sample was determined by comparing xylenecyanol concentrations based upon OD absorbance before and afterseparation. This percent recovery was then used to standardize theamount of solution added to the PCR reaction. Based on absorbancemeasurements, it was determined that recoveries of up to 95% with40-fold concentration were possible even in this gel box-based system.Higher recoveries and greater sample concentration can be expected asapplication specific electrophoresis units are employed.

Amplification

Following purification from humic acid, bacterial genomic DNA wasamplified by PCR. PCR threshold cycle (Ct) values were determined andcompared to the Ct values of control samples that did not undergo thepurification method of the invention (FIGS. 7 and 8).

Isotachophoretic Size Exclusion of Large Molecular Weight Nucleic Acids

Small nucleic acids were not concentrated with the bacterial and viralnucleic acids because they migrated in the gel faster than the leadingedge of the fast moving electrolyte. Therefore, there was a sizeseparation component as well. Size is also a critical difference betweenhuman genomic DNA and bacterial/viral nucleic acids in that humangenomic nucleic acid is much larger. The concentration for cross-linkingis adjusted according to the size of nucleic acid desired to beisolated.

In order to test this hypothesis, calf thymus DNA was ordered from Sigmachemical company along with a high molecular weight DNA ladder.Traditionally, DNA ladders consist of DNA fragments of known sizes thatcan be run on a gel as a standard to estimate the molecular weight of anunknown nucleic acid tested in parallel. However, the ladder was usedherein to test whether or not large nucleic acid molecules could beseparated while at the same time concentrating nucleic acids with sizesin the molecular weight range of bacteria and viruses. The firstexperiment (FIG. 9) was performed according to the laboratory protocoldescribed in the provisional patent in which a 1% agarose gel wasprepared and run. The results of this experiment showed that most largemolecular weight DNA (both calf thymus and the large molecular weightDNA ladder) entered the gel and concentrated with nucleic acid in thesize range of bacteria and viruses. However, in a second experiment(FIG. 10), when a 2% agarose gel was tested under the same conditionsthe large molecular weight DNA remained in well and did not migrate intothe gel. A portion of the large molecular weight DNA in the ladderentered the gel, but could not keep up with the moving buffer front. Noequivalent-sized molecular weight DNA was observed in the calf thymusDNA sample. The calf thymus DNA sample did contain some DNA of theappropriate size that it concentrated in the same region that bacterialand viral nucleic acid would concentrate, but that might be removed byfurther optimizing gel concentrations to remove it. Regardless, thisexample demonstrated that significant amounts of large molecular weightnucleic acid could be separated from nucleic acid in a size rangeequivalent to bacteria and viruses by excluding the high molecularweight eukaryotic genomic nucleic acids from the isotachophoresis gel.Therefore, this provides a solution for the detection of bacteria andviruses using next generation detection approaches.

Example 2

Isotachophoresis agarose gels were prepared using the identicalequipment described above except the buffers employed in the separationprocess were changed. Separation and concentration of protein and DNAwas accomplished using TAPSO NaOH as a buffer in place of Bicine (FIG.11) as well as a buffer system that separates and concentrates bothnucleic acids and proteins using a dual buffer system (FIG. 12).

TABLE 1 TAPSO Buffer Run 1% Gel, (0.4M Bis-Tris, 0.05M MES) 0.301 gagarose 12 ml 1.0M Bis-Tris 3 ml 0.5M MES 15 ml filtered diH₂0 1.5 μlEtBr

The gel solution above was prepared and poured into the gel box. Oncesolidified, the wells were loaded with 12 μl each of xylene cyanol dye,500-10 Kb ladder standard, 7.24 μg/ul BG DNA, 1 mg/ml OVAL, 1 mg/ml BSA,and xylene cyanol dye. A TAPSO buffer (0.2 M TAPSO, 0.1 M NaOH) wasadded to the negative electrode side of the gel box and a Bis-Trisacetate buffer (0.4 M Bis-Tris, 6 mM acetic acid) was added to thepositive electrode side of the gel box. The buffers were poured untilthey reached the top surface of the gel without flowing over the top ofthe gel. The gel was run at 25 V for 3.5 hours.

TABLE 2 TAPSO-Bicine Buffer Run 1% Gel, (0.4M Bis-Tris, 0.05M MES) 0.308g agarose 12 ml 1.0M Bis-Tris 3 ml 0.5M MES 15 ml filtered diH₂0 1.5 μlEtBr

A gel solution was prepared and poured into the gel box. Oncesolidified, the wells were loaded with 12 μl each of xylene cyanol dye,500-10 Kb ladder standard, 7.24 μg/ul BG DNA (ran in 2 lanes), 1 mg/mlOVAL, 1 mg/ml BSA, and xylene cyanol dye. A TAPSO buffer (0.2 M TAPSO,0.1 M NaOH) was added to the negative electrode side of the gel box anda Bis-Tris acetate buffer (0.4 M BisTris, 6 mM acetic acid) was added tothe positive electrode side of the gel box. Buffers were poured untilthey reached the top surface of the gel without flowing over the top ofthe gel. The gel was run at 25 V for 30 minutes. The TAPSO buffer wasthen removed and replaced with a Bicine buffer (0.2 M Bicine, 0.1 MNaOH). The run continued at 25 V for another 2.5 hours.

FIG. 11 shows a clear spatial separation of the two proteins which ranat the TAPSO NaOH/Bis-Tris front (shown by the white line) from the DNA(BG), which ran above the buffer front. FIG. 12 shows a buffer system ofTAPSO NaOH/Bis-Tris MES/Acetic acid which was run for the first 30minutes of the run followed by the removal of TAPSO NaOH and itsreplacement with Bicine in the buffer chamber. This resulted in twobuffer fronts. This gel showed clear spatial separation of the twoproteins which ran at the TAPSO NaOH/Bis-Tris MES front (shown by solidline) from the DNA (BG) which ran at the Bicine/TAPSO NaOH front shownby the dashed line. These Figures clearly show that the TAPSO buffersystem allows separation and concentration of nucleic acids and proteinsto different locations so that they can be processed separately.

Example 3 Isolation of RNA and Proteins

293FT cells were grown overnight on supplemented DMEM culture media. Thegrowth media was removed from the culture flask and the cells werewashed twice with 10 mL of phosphate buffered saline (PBS). Cells werecollected from the flask by scraping into a final volume of 1 mL of PBSfollowed by centrifugation at 1000×g for 5 minutes to pellet the cells.A 500 μL volume of lysis buffer was then added to the cells with mixingby gently repeatedly pipetting the cells. The cells were placed at −80°C. for 5 minutes followed by a 2 minute incubation in a 37° C. waterbath. This freeze thaw procedure was performed 3 times. Cellular debrisin the extract was then concentrated by centrifugation at 1000×g for 2minutes and the supernatant was carefully transferred to a new tube andkept at 4° C.

RNA purification was performed using the ToTALLY RNA™ Kit from AmbionInc.

according to manufacturer's instructions. After extraction, theresulting pellet was suspended in RNA elution buffer and stored at −20°C. until used the following day.

Agarose gels were cast directly into MINISUB® gel GT gel boxes (Bio-RadLaboratories) and wells were formed using a 15 well comb. Voltageapplied to the gels was supplied from a BioRad Power Pac Universal. Gelswere visualized using a VersaDoc gel imager in conjunction with theQUANTITY ONE® computer program, both from BioRad. Protein gels werephotographed using a handheld Nikon E4300 camera.

Bicine buffer, TAPSO buffer, MES, sodium hydroxide, acetic acid,ethidium bromide xylene cyanol, coomassie blue, and methanol were allproducts of Sigma Aldrich. Agarose MP which is manufactured specificallyfor the preparation of low percentage gels allowing the separation ofhigh molecular weight nucleic acids was purchased from RocheDiagnostics. The DNA ladder used was BioRad EZ Load™ HT Molecular Marker500 bp-10 kb.

The Bicine anode buffer was comprised of 0.2 M Bicine and 0.1 M NaOH. A200 mL volume of a 1.0 M Bicine stock solution and 100 mL of a 1.0 MNaOH stock solution were added to 700 mL of deionized water to produceone liter of buffer. The TAPSO anode buffer was comprised of 0.2 M TAPSOand 0.1 M NaOH. One liter of buffer was prepared by adding 400 mL of a0.5 M TAPSO stock solution, and 100 mL of a I M NaOH stock solution to500 mL of deionized water. The BisTris cathode buffer was 0.4 M Bis-Trisand 6 mM acetic acid and was prepared by adding 400 mL of a 1.0 MBis-Tris solution and 6 mL of a 1.0 M Acetic Acid to 594 mL deionizedwater.

Gels were prepared in a solution containing 0.4 M Bis-Tris and 0.05 MMES. This buffer was prepared by combining 12 mL of a I M Bis-Tris stocksolution, 32 mL of a 0.5 M MES stock solution, and 15 mL deionizedwater.

Gels were prepared by adding 0.3 g of agarose to 30 mL of BisTris MESbuffer for a final gel concentration of 1% agarose (w/v). A 1.5 μLvolume of an ethidium bromide stock solution was added to the gel toallow post-run visualization of nucleic acids. After gels were pouredand hardened the anode and cathode buffers were added to the anode andcathode buffer chambers, respectively. Buffers were added so that theirheight was equal to the top of the gel, but did not spill over top ofthe gel. Therefore, the gel was run as a “sea level” gel as opposed to asubmerged gel to prevent the anode and cathode buffers from mixingduring the run. Cellular extracts and purified RNA concentrations wereadjusted visually based on previous runs to yield a concentration thatwas easily distinguishable by visualization with a camera. Thesesolutions were added to wells in the gel as shown in FIG. 13 and the topwas placed onto the gel box. The electrophoretic separation wasperformed at 25 volts (9 milliamps) for 3.5 hours. The gels were runslowly to prevent heating which causes slight changes in anode bufferisoelectric points. In some applications gels have been run at 125 V foras short at 20 minutes and shown excellent separation. (It is expectedthat the invention would also include run times that could be decreasedsubstantially by increasing the voltage for this application as well orby developing an apparatus specifically for this type of separation thatwould not require the use a conventional gel box.) After the run wascomplete, the gel was transferred to the VersaDoc gel imager forphotographic documentation of nucleic acid content followed by transferto an acetic acid fixative and coomasie blue staining for visualizationof protein separation.

In a purely isotachophoretic approach the gel matrix itself would bepoured at a low enough concentration that there would be no separationeffect due to molecular size. However, even using gels that allowmigration of high molecular weight DNA, the gels cannot be poured at alow enough concentration that would completely prevent molecular weightseparation and at the same time allow the gels to be handled forvisualization. Therefore, some degree of molecular weight separation inthis system cannot be avoided.

Therefore the molecular weight separation was exploited to allow a dualseparation common to 2-dimensional gel separation, but in only a singledimension. This allows the dual separation to be conductedsimultaneously with no manual intervention, which is critical for thedevelopment of a rapid sample preparation cartridge for integration intoan automated system or a stand-alone purification device.

The simultaneous dual separation approach allows benefits of both sizeand charge to be exploited along with the advantage of concentrationgained by isotachophoresis. A number of different cross-linkingpercentages were investigated it has been determined that by alteringgel concentration different size ranges of nucleic acids can be“windowed out”, while concentrating all nucleic acids that separatewithin that window to a single location. FIG. 13 shows anisotachophoresis gel with separated nucleic acids and proteins from a293FT human cell extract (lane 2) and purified RNA from the same extract(lane 3). Lane 1 contains molecular weight markers.

In the gel that was run in FIG. 13, a 1% agarose MP concentration wasused, although other gel concentrations could also be used. Lane 2contained the 293FT cell extract without purification. In this instance,the agarose cross-linking in the gel prevented genomic DNA from enteringthe gel matrix and it remains near the loading location. Near the centerof the gel are 2 bands that is possibly due to mitochondrial DNAs.Mitochondrial DNA is roughly the size of a bacterial genome and it hasbeen demonstrated previously, using a slightly differentisotachophoretic approach that these bands, along with microbialgenomes, can be concentrated and purified into a single location on thegel. Finally, cellular RNAs are concentrated at the TAPSO/MES bufferfront. In order to prove that the material at the TAPSO/MES buffer frontwas indeed cellular RNAs, a portion of the identical cell extract shownin lane 2 was extracted for total RNA and the resulting purified RNA(lane 3) was run side by side with the cell extract. The resultingpurified RNA ran to the identical location of the presumed RNA from thecell extract. The purified RNA also had a lighter band of lowermolecular weight that ran below the TAPSO/MES buffer front, which islikely due to RNA degradation. Other procedures, using an extractionstep that omitted the use of chloroform did not show the band.

Protein staining of the identical gel showed protein concentrated atboth the TAPSO/MES front and the Bicine/TAPSO front. Clearly, the totalpurification of RNA needs to exclude protein and although some proteinsare excluded from the RNA band resulting in a partial purification, aconsiderable amount of protein copurifies with the RNA. If removal ofthis protein content is critical, then a proteinase treatment similar tomany other purification schemes that would result in reducing proteinsto a small size so that they separate below the TAPSO/MES front in asimilar location to the degraded nucleic acid observed in the phenolchloroform extracted RNA (lane 3). Such a proteinase treatment would berelatively quick and peptide purification from RNA post-proteinasetreatment could likely be performed simultaneously with separation ofRNA from other nucleic acids.

The invention demonstrates that RNA can be separated from other nucleicacids present in a cell extract and simultaneously concentrated byisotachophoresis at the interface of a buffer front. As described above,it has previously been shown that simultaneous concentration andpurification of the bacterial genomic DNA and viral RNA using slightlydifferent conditions can be optimized for these purposes, but with thesame general approach and set-up. Therefore, an opportunity is createdto have a single automated system that runs slightly different programswith different sets of disposables for all of these purposes.

Having now fully described this invention, it will be understood tothose of ordinary skill in the art that the same can be performed withina wide and equivalent range of conditions, formulations, and otherparameters without affecting the scope of the invention or anyembodiment thereof. All patents and publications cited herein areincorporated by reference in their entirety.

What is claimed is:
 1. A kit for separating and concentrating nucleicacid and protein targets, the kit comprising: labeled reagents whichaffect simultaneous co-purification and concentration of a nucleic acidand a protein; a gel isotachophoresis separation unit to which a samplecomprising the nucleic acid and the protein is added, the gelelectrophoresis comprising a gel box including a negative electrode sideand a positive electrode side, the negative electrode side being filledwith a first buffer comprising2-Hydroxy-N-(tris(hydroxymethyl)methyl)-3-aminopropanesulfonic acidbuffer and the positive electrode side being filled with a second bufferbeing different than the first buffer, the gel isotachophoresisseparation unit being configured to subject the sample toisotachophoresis using a voltage applied to the gel electrophoresisunit; a detection unit for the detection of the presence of the nucleicacid and the protein; and instructions for use.
 2. The kit of claim 1,wherein the detection unit is configured to detect the presence of aprotein selected from the group consisting of immunoassay, proteinsequencing, mass spectrometry, functional assays, non-antibody ligandbinding, aptamers, gels, and combinations thereof, and the detectionunit is further configured to detect the presence of a nucleic acidselected from the group consisting of PCR, isothermal amplificationmethods, hybridization reactions, microarrays, protein-DNA binding, massspectrometry, gels, and combinations thereof.
 3. The kit of claim 1,wherein the detection unit is configured to detect the presence thepresence of the nucleic acid and the protein in a single output.
 4. Thekit of claim 1, wherein the gel isotachophoresis separation unit isconfigured to simultaneously purify and concentrate both the nucleicacid and the protein by driving the nucleic acid toward the positiveelectrode side and driving the protein toward the negative electrodeside responsive to application of the voltage.
 5. The kit of claim 4,wherein the gel isotachophoresis separation is disposed in a handheldcartridge.
 6. The kit of claim 5, wherein the handheld cartridge isdisposable.
 7. The kit of claim 1, further comprising one or moredetection units comprising cartridges that would all run on a single,portable instrument.
 8. The kit of claim 1, wherein the isotachophoresisseparation unit further comprises a solidified gel matrix including atop surface, the negative electrode side being filled with the firstbuffer to a first buffer fill level being equal to or below the topsurface of the solidified gel matrix and the positive electrode sidebeing filled with the second buffer to a second buffer fill level beingequal to or below the top surface of the solidified gel matrix.